Williams Hematology



Abnormalities of the Signal Mechanism as a Result of Antibody or Complement Defects
Abnormalities of the Cellular Responses as a Result of Defects in Cytoplasmic Movement

Degranulation Abnormalities

Adhesion Abnormalities

Neutrophil Actin Dysfunction

Disorders of Neutrophil Motility

Other Disorders of Neutrophil Motility

Hyperimmunoglobulin e Syndrome
Defects in Microbicidal Activity

Chronic Granulomatous Disease

Myeloperoxidase Deficiency

Deficiencies of Glutathione Reductase and Glutathione Synthetase
Diagnostic Approach to the Patient with Suspected Neutrophil Dysfunction
Chapter References

The differential diagnosis for a patient presenting recurrent infections is formidable, given the complexity of the immune system. The clinical presentation of a patient who has a qualitative neutrophil abnormality may be similar to that of one who has an antibody or complement disorder. In general, evaluation for phagocyte cell disorders (Table 72-1) should be initiated among those patients who have at least one of the two following clinical features: (1) two or more systematic bacterial infections; (2) frequent, serious respiratory infections, such as pneumonia or sinusitis, or frequent bacterial infections such as cellulitis, draining otitis media, or lymphadenitis; (3) infections present at unusual sites (liver or brain abscess); and (4) infections associated with unusual pathogens (e.g., Aspergillus pneumonia, disseminated candidiasis, or infections with Serratia marcescens, Nocardia species, and Burkholderia cepacia).

Acronyms and abbreviations that appear in this chapter include: cAMP, cyclic adenosine monophosphate; CDP, CCAAP displacement protein; CGD, chronic granulomatous disease; CHS, Chédiak-Higashi syndrome; EBV, Epstein-Barr virus; FMF, familial Mediterranean fever; GDP, glucose diphosphate; G-6-PD, glucose-6-phosphate dehydrogenase; HLA, human leukocyte antigen; Ig, immunoglobulin; INF, interferon; LAD, leukocyte adhesion deficiency; LFA-1, lymphocyte function-associated antigen 1; MBP, mannose-binding protein; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NBT, nitroblue tetrazolium; PCR, polymerase chain reaction; phox, phagocyte oxidase; rINF, recombinant interferon; SGD, specific granule deficiency; SH3, SRC homology 3.


Neutrophils serve as the first line of defense against most bacterial pathogens. This function requires that the host have sufficient numbers of neutrophils that respond to chemotactic stimuli and ingest and kill bacteria. Chronic granulomatous disease (CGD) of childhood was the first qualitative neutrophil abnormality described. In this disorder, neutrophils are capable of ingesting but not killing certain microorganisms.
Neutrophil dysfunction may arise from (1) absence of antibodies or complement components required to opsonize microorganisms, an interaction that provides a chemotactic signal; (2) abnormalities of cytoplasmic movement that alter the chemotactic response or that result in abnormalities of the plasma membrane affecting the cell’s intrinsic capability to modulate movement; or (3) defects in microbicidal capability. Other comprehensive reviews of these syndromes are available to the interested reader.1,2,3 and 4
Since the synergistic action of immunoglobulins and complement proteins creates the opsonins that coat microorganisms and stimulate the development of chemotactic factors, a deficiency of either one may result in impaired neutrophil function. The most profound disturbances arise from abnormalities in C3, since this protein is the focal point for generation of opsonins and chemotactic factors (see Chap. 5).5,6 Activation of C3 can occur in the absence of either antibody or the classical complement components, C1, C4, and C2; thus, disorders of these molecules result in less severe clinical conditions. C3 deficiency is inherited as an autosomal recessive disorder.5 Homozygotes have undetectable serum levels of C3 and suffer from recurrent severe pyogenic infections, while asymptomatic heterozygotes have half the normal values.
A functional deficiency in C3 protease resulting in severe pyogenic infections also is seen in patients with a deficiency in C3b inactivator, a protein inhibitor of the alternative complement pathway. Unchecked activation of this pathway leads to hypercatabolism of C3 and factor B.7 Properidin deficiency also results in a functional deficiency in C3.8 Properidin is a serum protein that belongs to the alternative complement pathway; it is involved in the stabilization of the enzyme complex C3b22Bb. The protein is a multimeric glycoprotein with a subunit Mr of 56,000, the gene for which has now been cloned.9 Absence of properidin is associated with severe, often fatal pyogenic infection, often with meningococci.
Approximately 5 percent of the population have low serum levels of mannose-binding protein (MBP).10 It is a serum lectin secreted by the liver that binds mannose sugars present on the surface of bacteria, fungi, and some viruses. Mannose-binding protein is one of the collectin-soluble effector proteins that contribute to the basic armamentarium of nonclonal immunity. Mannose-binding protein can function as an opsonin when bound to surfaces by activating the complement cascade. A deficiency of MBP has been reported in infants with frequent unexplained infections, chronic diarrhea, and otitis media.10 Deficiency of MBP in adults with recurrent infections has also been associated with autosomal dominant inheritance of point mutations of MBP polypeptide, which leads to the failure of MBP to activate complement.11,12 and 13 It is possible that the adults expressing the defective MBP are lacking the ability to produce anticarbohydrate antibodies, which further predisposes them to recurrent infections.
Because of the large number of chemoattractants generated during inflammation, it is difficult to establish the relative significance of a given individual component. Furthermore, chemotactic factors and opsonins are involved in the activity of both neutrophils and mononuclear phagocytes. Therefore, it is not clear whether the clinical consequences of disorders involving these substances are unique to one or the other of these phagocytic cells. Patients with antibody- or complement-deficiency syndromes suffer mainly from infections with encapsulated pathogens such as Haemophilus influenzae, pneumococci, streptococci, and meningococci.14 Furthermore, splenectomized individuals, deprived of an organ rich in mononuclear phagocytes, have a small but finite risk of sepsis due to these same microorganisms.15 Encapsulated pathogens characteristically are not associated with neutropenic states. Antibody coating of encapsulated organisms facilitates their ingestion by mononuclear phagocytes but may be less important for their ingestion by neutrophils.
Definition and History This rare autosomal recessive disease was initially recognized as one in which neutrophils, monocytes, and lymphocytes contained giant cytoplasmic granules (Fig. 72-1). Chédiak-Higashi syndrome (CHS) is now recognized as a disorder of generalized cellular dysfunction characterized by increased fusion of cytoplasmic granules.16 Pigmentary dilution affecting the hair, skin, and ocular fundi results from pathologic aggregation of melanosomes and is associated with a failure of decussation of the optic and auditory nerves (Table 72-1).17 Patients with this syndrome exhibit an increased susceptibility to infection, which can be explained at least in part through defects in neutrophil chemotaxis, degranulation, and bactericidal activity. The presence of giant granules in the neutrophil interferes with their ability to traverse narrow passages between endothelial cells. Other features of the disease include neutropenia, thrombocytopathy, natural killer cell abnormalities, and peripheral neuropathies.18 Similar genetic syndromes have been described in mice, mink, cats, rats, cattle, and killer whales.19

FIGURE 72-1 Blood films of patients with the Chédiak-Higashi syndrome. (a) The granulocyte contains large amorphic cytoplasmic granulations. (b) A large inclusion is easily seen in a lymphocyte.

Etiology and Pathogenesis Although the basic mechanism underlying CHS is unknown, alterations in membrane fusion probably play an important role.16,20,21 and 22 It appears CHS is caused by a fundamental defect in granule morphogenesis that results in abnormally large granules in multiple tissues. Giant granules are seen in Schwann cells, leukocytes, and macrophages of the liver and spleen, and certain cells of the pancreas, gastric mucosa, kidney, adrenal gland, and pituitary gland. Giant melanosomes form and prevent the even distribution of melanin, which results in pigmentary dilution of the hair, skin, iris, and optic fundus. In the early stages of myelopoiesis some of the normal-size azurophil granules coalesce to form giant granules that result in large secondary lysosomes that contain reduced content of hydrolytic enzymes, including proteinases, elastase, and cathepsin G.16,23,24,25 and 26 Many of the myeloid precursors die in the marrow, resulting in a moderate neutropenia, with white cell counts of about 2500/µl (2.5 × 109/liter).27 In spite of the normal ingestion of particles and active oxygen metabolism, these neutrophils kill microorganisms relatively slowly. This delay reflects a slow and inconsistent delivery of diluted amounts of hydrolytic enzymes from the giant granules into the phagosomes, which may predispose the host to bacterial infection.16,20 In this syndrome, monocytes have the same functional derangements as neutrophils.16
The CHS blood cell membranes are more fluid than cells of normal individuals,16,22 and the altered membrane structure could lead to defective regulation of membrane activation. Conceivably, changes in membrane fluidity may affect cell function by altering expression of membrane receptors. This, in turn, could result in elevated levels of intracellular cyclic adenosine monophosphate, disordered assembly of microtubules, and the defective interaction of microtubules with lysosome membranes, which occur in this disorder and are reflected in the reduced chemotactic responses.16
The gene for CHS, known as LYST, has been cloned based on its homology to the murine gene responsible for mouse CHS (beige phenotype).28 The gene was recognized by the presence of LYST mutations. The gene is localized on chromosome 1q42–q44 and has structural features homologous to a vacuolar sorting protein called VPS15 in yeast. The CHS protein may be associated with vacuolar transport and mediate protein-protein associations that integrate cellular signal response coupling.
Clinical Features Characteristically patients with CHS have light skin and silvery hair. They frequently complain of solar sensitivity and photophobia. Other signs and symptoms vary considerably. Infections and neuropathy are common. The infections involve the mucous membranes, skin, and respiratory tract. They are susceptible to both gram-positive and gram-negative bacteria as well as fungi, with Staphylococcus aureus being the most common infecting organism. The neuropathy may be sensory or motor in type, and ataxia may be a prominent feature.
Patients with CHS have prolonged bleeding times with normal platelet counts, resulting from impaired platelet aggregation associated with a deficiency of the storage pools of adenosine diphosphate and serotonin.29 Natural killer cell function also is impaired.16,30 The diagnosis is established by the presence of large inclusions in all nucleated blood cells. These can be seen on Wright-stained blood films but are accentuated by peroxidase stains.
The accelerated phase of CHS is characterized by lymphocytic proliferation in the liver, spleen, and bone marrow. The accelerated phase may occur at any age. Typically the patient develops hepatosplenomegaly and high fever in the absence of bacterial sepsis. The pancytopenia becomes worse at this stage, producing hemorrhage and an increased susceptibility to infection. The onset of the accelerated phase may be related to the inability of these patients to contain and control the Epstein-Barr virus (EBV) and leads to features simulating viral-mediated hemophagocytic syndrome (see Chap. 5).31,32 The lymphocyte proliferation is associated with recurrent bacterial and viral infections, fever, and prostration, usually resulting in death. At autopsy, the lymphohistiocytic infiltrates in the liver, spleen, and lymph nodes are extensive, but not neoplastic by histopathologic criteria.16,30 Occasionally, giant lysosomes resembling those of CHS may be observed in acute myelogenous leukemia.33
Therapy, Course, and Prognosis High-dose ascorbic acid (200 mg/day for infants, 2 g/day for adults) has been found to improve the clinical status of some patients in the stable phase.16,34 Although there is controversy regarding the efficacy of ascorbic acid, given the safety of the vitamin it is reasonable to administer it to all patients. The CHS presents a therapeutic dilemma, particularly when the accelerated phase begins. Prophylactic antibiotics do not prevent infections. Treatment regimens with glucocorticoids and vincristine therapy have been tried, but their efficacy is not established.16 The only curative therapy for the accelerated phase is marrow transplantation from an HLA-compatible donor or an unrelated donor compatible at the D locus.35,36 Marrow transplantation constitutes normal hematopoietic and immunologic function and corrects the natural killer cell deficiency in patients entering the accelerated phase.36 Ocular and cutaneous albinism are not corrected after transplantation. Whether transplantation will prevent the neuropathies from developing remains to be determined. Development of a vaccine against EBV could delay or prevent the accelerated phase.
Specific granule deficiency (SGD) has been described in five patients of both sexes and is likely inherited as an autosomal recessive disorder (see Table 72-1).16 Besides the absence of specific granules, the nuclei of the neutrophils are bilobed. Patients are afflicted with recurrent infections primarily involving the skin and lungs. Staphylococcus aureus has been the most commonly observed pathogen, although Candida albicans and a variety of gram-negative bacteria also have been isolated. Specific granule–deficient neutrophils lack gelatinolytic activity in the tertiary granules; vitamin B12-binding protein, lactoferrin, and collagenase in the specific granules; and defensins in the primary granules.25,37,38 This disorder also extends to eosinophils that lack the characteristic eosinophil granule proteins, major basic proteins, eosinophilic cationic proteins, and eosinophil-derived neurotoxins (see Chap. 68).39 Thus, the disorder is a global defect in phagocytic granules rather than limited to specific granules, as suggested by its name. Neutrophils from these patients have abnormal chemotaxis, possibly related to the absence of the intracellular pool of leukocyte adhesion molecules that normally reside in the specific granules,40,41,42 and 43 and a mild defect in bactericidal activity, possibly related to the deficiency of the granule constituents lactoferrin and defensins.25,40 The impairment in granule protein synthesis affecting the granulocytic cells likely reflects a primary defect in gene expression, possibly in a shared transcription factor common to the affected subset of proteins.38 Such a transcription factor might be the CCAAP displacement protein (CDP) that binds to a specific region of both the lactoferrin and the collagenase promoter.44,45 This protein is the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil-specific granule protein genes. Overexpression of CDP leads to the coordinate loss of specific granule protein expression; thus, an abnormality in expression of CDP could account for the biochemical phenotype seen in SGD. Alternatively in one patient a five-basepair deletion in the transcription factor CCAAT/enhancer binding protein (C/EBP) epsilon, which is a factor known to regulate myeloid granule formation, was found.45 The defect is restricted to blood cells, since normal lactoferrin secretion has been demonstrated in the nasal secretions of a SGD patient despite the abnormality demonstrated in his neutrophils.37
The diagnosis of SGD is suggested by the presence of neutrophils devoid of specific granules but containing azurophilic granules on the blood film.16 Electron microscopy reveals small peroxidase-negative vesicles presumably representing empty specific granules.46 The diagnosis can be confirmed by demonstrating a severe deficiency in either lactoferrin or vitamin B12-binding protein. An acquired form of SGD can be observed in thermally injured patients or in individuals with myelodysplasia.16,47 Treatment of SGD is symptomatic, with the administration of parenteral antibiotics for acute infections and surgical drainage of refractory infections.16 With aggressive medical management, patients may survive into their adult years.
Definition and History Leukocyte adhesion deficiency type I (LAD 1) is a rare autosomal recessive disorder of leukocyte function. About 75 cases have been reported worldwide. The disease is characterized clinically by recurrent soft-tissue infections, delayed wound healing, and severely impaired pus formation despite striking blood neutrophilia.3,48 Individuals with this disorder have a decreased or absent expression of a family of structurally and functionally related leukocyte surface glycoproteins designated CD11/CD18 complex (also referred to as the b2-integrin family of leukocyte adhesive proteins; see Table 72-1). These proteins include LFA-1 (CD11a/CD18), Mo-1 or Mac-1 (CD11b/CD18), and p150,95 (CD11c/CD18).48 The CD11 subunits are integral membrane glycoproteins, each spanning the plasma membrane only once. They are approximately 40 percent homologous, suggesting that they arise from a common primordial gene.48 The three distinct genes encoding the a subunits occur in a cluster on chromosome 16, whereas the gene for the b subunit is located on chromosome 21.49
Etiology and Pathogenesis Each of these molecules contains an a and a b subunit noncovalently associated in an ab structure. They all have the same b subunit and are distinguished by their a subunits, which have different isoelectric points, molecular weights, and cell distribution (Table 72-2).48 The structure of CD11/CD18 has been deduced from molecular cloning of the various subunits.50,51 and 52 These studies have established that CD11/CD18 are members of a large gene family involved in cell-cell and cell-matrix adhesion (integrins).53 Several subfamilies of integrins have been described and classified according to the type of their highly homologous b subunits.54 The a subunits are also homologous to each other, but to a lesser degree than are the associated b subunits. Within each subfamily, a single b subunit usually is shared by several a subunits. Certain a subunits often share more than one b subunit, which alters their specificity for various ligands.54 The molecular defect involves all three members of the CD11 integrin subfamily. In the patients with LAD I who have been evaluated at the molecular level, absent, diminished, or structurally abnormal b subunits (CD18) have been identified. A heterogeneous group of mutations that are confined to the gene on chromosome 21q22.3 have been identified.55 Many patients have point mutations that result in single amino acid substitutions in CD18, which predominantly reside between amino acids 111 and 361.56,57,58,59,60,61 and 62 This peptide domain is highly conserved among all b subunits and appears to be important for interaction with the a subunit. Several affected individuals are compound heterozygotes for two different mutant alleles, whereas others are homozygotes for a single mutant allele. Messenger RNA splicing abnormalities described in two kindreds can result in either deletion or insertion of amino acids in the conserved extracellular domain of CD18.63 Small deletions within the coding sequences of the CD18 gene disrupting the reading frame or a nucleotide substitution resulting in a premature termination signal has been described.57,62,65 Mutations in CD18 disrupt the association in the ab subunits so that maturation, intracellular transport, and cell surface assembly of functionally active ab molecules fail to occur.48 Approximately half of patients exhibit a low level of CD11/CD18 cell surface molecules and moderate disease, with the remainder having totally absent surface expression of these proteins, which accounts for a profound impairment of neutrophil and monocyte adherence and adhesion-dependent functions in vitro, including cell migration, phagocytosis, and complement- or antibody-dependent cytotoxicity.66,67


Besides the requirement for surface expression of the CD11/CD18, the molecules must undergo posttranslational modification during leukocyte activation.68 In one patient with LAD, the integrin molecules were expressed on the surface of the neutrophil but failed to undergo high-avidity ligand binding.69 The resulting neutrophil functional abnormalities resulted in a moderate clinical disorder in the patient. The bulk of the neutrophil Mac-1 glycoprotein is stored inside the cell in the membrane of neutrophil-specific gelatinase and secretory granules.70,71 and 72 Exposure of neutrophils to degranulating stimuli results in a five- to tenfold increase in the number of Mac-1 molecules on the cell surface, which parallels the fusion of granules to the plasma membrane.71 Neutrophils from these patients fail to augment their surface adhesive glycoproteins, since the defect in b-subunit synthesis affects both membrane and granule pools of Mac-1.73 In contrast to Mac-1 and p150,95, lymphocyte function-associated antigen 1 (LFA-1) is predominantly confined to the neutrophil plasma membrane. Consequently, the cell surface levels of LFA-1 are not enhanced by neutrophil degranulation.
Lymphocytes deficient in CD11/CD18 are able to adhere to endothelial surfaces via the expression on lymphocytes of very late activation 4 (VLA-4) integrin receptors, which bind to the vascular cell adhesion molecule 1 (VCAM-1), found on the endothelial cells74; this residual adhesion may account for the paucity of clinical symptoms related to lymphocyte function.
The failure of the leukocyte adhesion–deficient neutrophils to migrate to the sites of inflammation outside of the lung and peritoneum is due to their inability to adhere firmly to surfaces and undergo transendothelial migration from venules.75,76 and 77 Failure of CD11/CD18-deficient neutrophils to undergo transendothelial migration occurs because b2-integrins bind to intercellular adhesion molecules 1 and 2 (CD54 and ICAM-2) expressed on inflamed endothelial cells.48,78 The neutrophils that do arrive at inflammatory sites in the lung and peritoneum by CD11/CD18-independent processes fail to recognize micro-organisms coated with the opsonic complement fragment C3bi (an important stable opsonin formed by the cleavage of C3b by C3b inactivator).48,53 Other neutrophil functions such as degranulation and oxidative metabolism normally triggered by C3bi binding are also diminished and markedly compromised in neutrophils from LAD 1.48 Similarly, the urokinase-plasminogen activator-receptor and the FcgRIII receptors, both phosphatidylinositol-linked proteins, are defective in their functions because these receptors transduce their signals through CD11/CD18.79,80 Monocyte function is also impaired. Monocytes of affected individuals have poor fibrinogen-binding function, an activity promoted by the CD11/CD18 complex48,81; consequently, such cells are not able to participate effectively in wound healing. Thus, impairment in neutrophil function underlies the propensity to have recurrent infections, which is the clinical expression of this disease. Similar genetic syndromes have been discovered in a dog and Holstein cattle.82,83 A CD11/CD18-deficient mouse with 2 to 6 percent of normal b2-integrin expression has been produced by gene targeting.75,84
Clinical Features Activated leukocytes of patients with the most severe clinical form express less than 0.3 percent of the normal amount of the b2-integrins, whereas those of patients with the moderate phenotype may express 2 to 7 percent of normal numbers of b2-integrin molecules.48 The severely affected patients suffer from recurrent and chronic or even gangrenous soft-tissue infections (subcutaneous tissues or mucous membranes), generally by bacterial or fungal microorganisms such as Staph. aureus, Pseudomonas spp. and other gram-negative enteric rods, or Candida spp. Patients with the moderate phenotype have fewer and less severe infections. Infectious susceptibility and impaired wound healing are related to diminished or delayed infiltration of neutrophils and monocytes into extravascular inflammatory sites. In all patients surviving infancy, severe progressive generalized periodontitis is present. Individuals who are clinically well but are heterozygous carriers of LAD have been identified. Their stimulated neutrophils express approximately 50 percent of the normal amount of the Mac-1 a subunit and the common b subunit.48
The diagnosis of LAD is suggested by one or more clinical features, including recurrent cutaneous, periodontal, or other soft-tissue infections, as well as delayed wound healing and delayed umbilical cord severance and/or infections, especially in the setting of persistent neutrophilia.
Laboratory Features The diagnosis is made most readily by flow cytometric measurement of surface CD11b in stimulated and unstimulated neutrophils using monoclonal antibodies directed against CD11b (Fig. 72-2). Assessment of neutrophil and monocyte adherence, aggregation, chemotaxis, C3bi-mediated phagocytosis, and cytotoxicity generally will demonstrate striking abnormalities that are directly related to the molecular deficiency. Delayed-type hypersensitivity reactions are normal, and most individuals have normal specific antibody synthesis. The ability of lymphocytes to generate specific antibodies explains the self-limited course of varicella or viral respiratory infections. However, some patients have impaired T-lymphocyte–dependent antibody responses, for example, to repeat vaccination with tetanus toxoid, diphtheria toxoid, and polio virus.

FIGURE 72-2 Specific diagnosis of CD11/CD18 glycoprotein deficiency by indirect immunofluorescence flow cytometric analysis. Blood neutrophils of a pediatric patient suspected of having CD11/CD18 glycoprotein deficiency and those of an abnormal individual were subjected to immunofluorescence staining for the expression of the CD11b, CD11a, CD11c, and CD18 epitope (cross-hatched histogram) as compared with the background immunofluorescence staining by isotype-identical negative-control antibodies (open histograms). Neutrophils were either stained immediately after purification by Ficoll-Hypaque density centrifugation (unstimulated) or after exposure to calcium ionophore A23187 (1 mM) for 15 min at 37°C (A23187-stimulated). A23187 stimulation causes significant increase in CD11b and CD18 epitope staining (surface MO1 expression) by normal neutrophils as compared with unstimulated normal cells. A23187 stimulation also causes a small increase in the CD11b-epitope expression of patient cells (the CD11b cross-hatched histogram becomes distinguishable from background staining after A23187 stimulation), suggesting that this patient has a “moderate” form of the disorder (capable of expressing small but detectable quantities of CD11/CD18 glycoproteins). Flow cytometric analysis was performed on a Coulter Electronics EPICS F C Flow Cytometer with a logarithmic amplifier. (From Todd and Freyer,202 with permission.)

Patients with LAD I usually have blood neutrophil counts of 15 to 60 × 109/liter. However, during infectious episodes, they commonly have neutrophil counts in excess of 100 × 109/liter and sometimes as high as 160 × 109/liter. Granulocytic hyperplasia is a feature of the marrow examination. Despite elevated blood counts, there is a paucity of neutrophils in inflammatory skin windows and biopsies of infected tissues.
Differential Diagnosis Two patients have been described who had neutrophilia, recurrent bacterial infections, and an inability to form pus.85 Both patients also had the Bombay blood phenotype and were the progeny of consanguineous parents, suggesting an autosomal recessive inheritance pattern. Functionally, the neutrophils were unable to adhere to E-selectin or cytokine-activated endothelial cells and exhibited impaired chemotaxis and an inability to roll on postcapillary venules in vivo. The patients also exhibited distinctive facial appearance, were short in stature, had severe mental retardation, and were secretor-negative and Lewis antigen–negative. These patients are now classified as having LAD II. In contrast to LAD I, the patient’s natural killer cell activity was normal.86 The LAD II neutrophils expressed normal levels of CD18 integrins but were deficient in the carbohydrate structure sialyl–Lewis X, which renders the cells unable to roll on activated endothelial cells expressing E-selectin. Thus, the neutrophils from the patients categorized as having LAD II are unable to tether to inflamed venules, which is necessary for subsequent activation (see Chap. 66). The basis for LAD II cells appears to be a defect in the de novo pathway of glucose diphosphate (GDP)-fucose biosynthesis, which leads to deficient formation of Fuca1phosphate ® 2 Gal linkages in ABO blood group core antigen, thereby accounting for the Bombay phenotype, whereas the Lewis antigen–negative phenotype arises from failure to synthesize Fuca1phosphate ® 4 GlcNAc and Fuca1phosphate ® 3 GlcNAc moieties.85,87
Therapy, Course, and Prognosis Treatment of this disorder is largely supportive.4,48,73 Patients with a history of recurrent infections can be maintained on prophylactic trimethoprim-sulfamethoxazole. Marrow transplantation with HLA-compatible siblings or parental donors has resulted in engraftment and restoration of neutrophil function88 and remains the treatment of choice for patients with a severe phenotype.
The restoration of CD11/CD18 expression in CD34 peripheral stem cells from LAD I following transduction with a retrovirus bearing CD18 and induced to differentiate into neutrophils with growth factors indicates that LAD I is caused by defective CD18 gene and provides a basis for somatic gene therapy.89 Not only did the neutrophils express the integrins, but the cells demonstrated improvement in their functional responses, such as adhesion and the respiratory burst when challenged with ligands for CD11/CD18. These results indicate that ex vivo of the transfer gene for CD18 into LAD I CD34+ cells followed by reinfusion of the transfused cells may represent a therapeutic approach to LAD.
The severity of infectious complications correlates with the degree of b2 deficiency.73 Patients with severe deficiency may die in infancy, and those surviving infancy have a susceptibility to severe life-threatening systemic infections. In patients with moderate deficiency, life-threatening infections are infrequent and survival relatively long.73 Fetal blood sampling and flow cytometric analysis for expression of CD11/CD18 integrins can be used for prenatal diagnosis of LAD I.90 However, contamination of the sample by maternal blood can complicate the interpretation of the analysis.
These infants, like patients with LAD, have recurrent pyogenic infection from birth as a result of defective chemotactic and phagocytic response (see Table 72-1). In one patient, actin isolated from blood neutrophils did not polymerize under conditions that fully polymerized the actin of neutrophils from normal individuals.91 Subsequent studies on the index patient’s family confirmed that partial actin dysfunction was present in the parents and one sister.92 One of the parents was found to be a heterozygote for LAD, but the other was not.93 Further studies established that LAD is not generally associated with defective actin filament assembly.94 The basis of the defective polymerization of actin in the index patient remains unknown, but this disorder of phagocytes is distinct from LAD.
Defective actin polymerization has been described in a 2-month-old infant with severe recurrent bacterial infections associated with impaired chemotaxis and phagocytic response.95 This patient’s neutrophils showed increased expression of CD11b, distinguishing the patient’s clinical problem from LAD I. Morphologically the neutrophils display thin, filamentous projections of membrane with an underlying abnormal cytoskeletal structure. Subsequently a 47-kD protein was purified that inhibited actin polymerization in vitro.96 Further biochemical studies revealed a markedly defective actin polymerization in the patient’s neutrophils along with a severe deficiency of an 89-kD protein and an elevated level of the 47-kD protein. The 47-kD protein has been identified as LSP-1 (the lymphocyte-specific protein–1), which is an actin-binding protein present in normal neutrophils. Overexpression of the LSP-1 has resulted in bundling of actin in cells, leading to an abnormal cytoskeletal structure and motility defects.97 Because actin dysfunction is lethal, treatment requires restoration of normal neutrophil function by marrow replacement from a normal donor. Bone marrow transplantation was attempted in both infants. In the first infant it was unsuccessful, whereas in the patient with the neutrophil actin dysfunction associated with overexpression of the 47-kD protein, bone marrow transplantation was successful.95,98
Definition and History Familial Mediterranean fever (FMF) is an autosomal recessive disease that primarily affects populations surrounding the Mediterranean basin. The disease is characterized by acute limited attacks of fever often accompanied by pleuritis, peritonitis, arthritis, pericarditis, inflammation of the tunica vaginalis of the testes, and erysipelas-like skin disease (see Table 72-1).
Etiology and Pathogenesis The pathologic findings in FMF are those of nonspecific acute inflammation affecting serosal tissues such as the pleura, peritoneum, and synovium. Neutrophilic infiltration predominates in the affected tissues. Physical and emotional stress, menstruation, and a high-fat diet may trigger the attacks.99
There is a lack of a C5a inhibitor activity in joints and peritoneal fluid in FMF, and since C5a is a highly potent chemotattractant for neutrophils, it has been suggested that lack of the inhibitor might account for the acute attacks of inflammation.100 This hypothesis has not been confirmed. The observation that FMF has clinical manifestations similar to those of systemic lupus erythematosus suggests the possibility of an underlying autoimmune disorder. However, FMF does not respond to steroids and autoantibodies have not been found.99,101
The gene responsible for FMF has been identified to be located on chromosome 16. It encodes for a 781–amino acid protein called pyrin.102,103 Homology searches indicate that pyrin is a new member of the RETRO gene family and suggests that pyrin itself may be a transcription factor, presumably regulating the expression of target genes, at least some of which are likely involved in the suppression of inflammation. The gene is expressed in neutrophils, but not other leukocytes. Pyrin has been designated as the gene for FMF because missense mutations have been identified in exon 10 in most of the affected patients, but not in normal subjects. Additional mutations in exon 2 of the gene have recently been detected in several families from various ethnic groups.99 These mutations have not been found in all patients, indicating that other mutations are likely to be discovered. It is possible that pyrin may be involved in attenuating neutrophil activation by chemotactic factors. A puzzle, however, remains as to why the serosal tissues are the main targets of inflammation in FMF.
Clinical Features The duration and frequency of attacks may vary considerably even in the same patient.99 Acute attacks frequently last 24 to 48 h and recur once or twice a month. In some patients, attacks may recur as frequently as several times a week or as infrequently as once a year, and symptoms may persist as long as a week during individual episodes. Some patients experience spontaneous remission that persists for years followed by recurrence of frequent attacks. Peritonitis due to FMF may resemble an acute abdomen, thereby leading to potential uncertainties about the clinical management of the acute abdominal episode. Attacks of pleuritic pain occur in about 25 to 80 percent of patients. Symptoms of pleuritis may sometimes precede abdominal pain, and some patients experience pleuritic attacks without abdominal symptoms. Recurrent pericarditis has been reported, rarely. The course of peritonitis in FMF is similar to attacks at other serosal sites; however, it tends to appear at a late stage of the disease. Mild arthralgia is a common feature of febrile attacks, and monoarticular or oligoarticular arthritis may occur. Arthritis usually affects large joints, the knees in particular, and effusions are common. As many as one-third of the patients experience transient erysipelas-like skin lesions that appear typically on the lower leg, ankle, or dorsum of the foot. These lesions are circumscribed, painful, erythematous areas of swelling, which usually subsides within 24 to 48 h.
In about 25 percent of affected patients a form of renal amyloidosis develops in which the amyloid derives from a normal serum protein called serum amyloid A (amyloidosis of the AA type; see Chap. 105). The amyloidosis progresses over a period of years to renal failure in almost all cases, and the cause of death in patients with FMF is usually attributed to this complication.
Laboratory Features Laboratory findings in FMF are nonspecific. During acute attacks leukocytosis (up to 30,000/ml) is present, and the erythrocyte sedimentation is increased. Between attacks the leukocyte count is normal.100
The cloning of the FMF gene now allows a reliable diagnostic test. By employing a set of polymerase chain reaction (PCR) primers, it is possible to identify the mutations responsible for the disease. Three major mutations are present in 85 percent of FMF carrier chromosomes. If the carrier gene frequency is 1 in 8, 98 percent of FMF patients will carry one or two of these mutations, and only 2 percent will bear an unidentified mutation.99
Therapy, Course, and Prognosis Colchicine treatment is effective in FMF and may prevent the development of amyloidosis.100 Prophylactic colchicine, 0.6 mg orally, two to three times a day, prevents or substantially reduces the acute attacks of FMF in most patients. Some patients can abort attack with intermittent doses of colchicine beginning at the onset of attacks (0.6 mg orally every hour for 4 h, then every 2 h for four doses, and then every 12 h for 2 days). In general, patients who benefit from intermittent colchicine therapy are those who experience a recognizable prodrome before developing fever and clear-cut acute symptoms.
The prognosis for normal longevity for patients has been excellent since the recognition of colchicine efficacy in this disease. Most patients can be maintained almost entirely symptom free. However, if amyloidosis develops, it may be followed by the nephrotic syndrome or uremia. Unless the patient receives a renal transplant, the likelihood of eventual death from renal failure is high.
The directed migration of neutrophils from the circulation to an inflammatory site is a consequence of chemotaxis and leads to the accumulation of an exudate. For normal chemotaxis to occur, a complex series of events must be coordinated. Chemotactic factors must be generated in sufficient quantities to establish a chemotactic gradient. The neutrophils must have receptors for the chemotactic agents and mechanisms for discerning the direction of the chemotactic gradient. Depressed neutrophil chemotaxis has been observed in a wide variety of clinical conditions (see Table 72-1).1 These can be stratified as follows: (1) defects in the generation of chemotactic signals; (2) intrinsic defects of the neutrophil; and (3) direct inhibitors of neutrophil motility in response to chemotactic factors.
Older patients with chemotactic disorders may be infected by a variety of microorganisms, including fungi and gram-positive or gram-negative bacteria.1 Staphylococcus aureus is the most frequent bacterial offender. Typically, the skin, gingival mucosa, and regional lymph nodes are involved. Respiratory tract infections are frequent, but sepsis is rare. Delayed or inappropriate signs and symptoms of inflammation are common. Although the cells move slowly in Boyden chambers or other chemotactic assays, they do accumulate in sufficient numbers in inflammatory sites to produce pus. However, detection of patients with neutrophils that have profound defects in chemotaxis usually is accomplished through other phagocytic assays.
Patients with the hereditary deficiency of complement factors C3, C5, or properidin exhibit an increased incidence of bacterial infections because they are unable to form the chemotactic peptide C5a.104 The degree to which defective chemotaxis plays a role in C3 deficiency is unclear because opsonization and ingestion rates also are abnormal in these disorders. Frequently, chemotactic disorders are associated with other impaired neutrophil functions. For instance, both glycogen storage disease type 1b and myelokathexis are chemotactic disorders frequently associated with an absolute neutrophil count below 0.5 × 109/liter.105,106 Following restoration of a normal neutrophil count with granulocyte colony stimulating factor, the patients no longer are predisposed to recurrent bacterial infections in spite of a persistent chemotactic defect. Thus, a chemotactic defect observed in vitro does not correlate invariably with decreased resistance to bacterial infections in vivo.
Among the impaired defense mechanisms of the neonate is neutrophil chemotaxis, as demonstrated by the in vitro response of neonatal neutrophils to a variety of chemotactic factors.76 The impaired motility of the neonatal neutrophils in part arises from the diminished ability to mobilize neutrophil b2-integrins following neutrophil activation.107 Additionally, the neonatal neutrophil may have a qualitative defect in b2-integrin function, resulting in impaired neutrophil transendothelial migration for up to 1 month after birth.
Although many pharmacologic agents can influence neutrophil function, few drugs used in clinical medicine affect neutrophil behavior in vivo. Ethanol in concentrations that occur in human blood can inhibit neutrophil locomotion and ingestion.108 Glucocorticoids, especially at high and sustained doses, inhibit neutrophil locomotion, ingestion, and degranulation.109 Administration of glucocorticoids on alternate days does not interfere with neutrophil movement.110 Epinephrine does not have a direct affect on neutrophil adhesion.111 Cyclic adenosine monophosphate (cAMP), which is released from endothelial cells following exposure to epinephrine, can depress neutrophil adherence. Similarly, elevated cAMP levels following epinephrine administration may impair neutrophil adherence, leading to diminished neutrophil margination and apparent neutrophilia. Immune complexes, as seen in patients with rheumatoid arthritis or other autoimmune diseases, also can inhibit neutrophil movement by binding to neutrophil Fc receptors.1
The hyperimmunoglobulin E syndrome is a disorder characterized by markedly elevated serum IgE levels, chronic dermatitis, and serious recurrent bacterial infections.112 The skin infections in these patients are remarkable for their absence of surrounding erythema, leading to the formation of “cold abscesses.” The neutrophils and monocytes from patients with this syndrome exhibit a variable but at times profound chemotactic defect that appears extrinsic to the neutrophil.
Approximately 150 patients have been reported with this disorder. Both males and females have been affected. A familial occurrence in successive generations is suggestive of an autosomal dominant form of inheritance. The molecular basis for this syndrome remains unknown. Some believe that the immunologic basis of hyperimmunoglobulin E arises from insufficient suppressor T cells, which is manifested in part by reduced production of interferon (IFN)-a and tumor necrosis factor (TNF).113 The proposed T-cell defect could explain the hyperproduction of IgE and the abnormal antibody responses that have been documented in some patients in response to various vaccines.114 The predisposition to bacterial infections may arise from production of a chemotactic inhibitor released by mononuclear cells that inhibits normal neutrophil chemotaxis.115 Another mechanism thought to predispose patients to recurrent bacterial infections is that the generation of excessive amounts of IgE directed against Staph. aureus and other varieties of bacterial and fungal antigens may be at the expense of the generation of protective IgE antibodies against the same organisms.114
Hyperimmunoglobulin E may begin as early as 1 to 8 weeks of age. The syndrome is characterized by chronic eczematoid rashes, which are typically papular and pruritic.112 The rash generally involves the face and extensor surfaces of arms and legs; skin lesions are frequently sharply demarcated and usually lack surrounding erythema. By 5 years of age all patients have had a history of recurrent skin abscess formation and recurrent pneumonias, along with chronic otitis media and sinusitis. Patients may also develop septic arthritis, cellulitis, or osteomyelitis. The major offending pathogen is generally Staph. aureus. Other associated features include coarse facial features, manifested by a broad nasal bridge, prominent nose, and irregularly proportional cheeks and jaw. Growth retardation is also found in a minority of patients and appears related to the presence of chronic illness. Occasionally osteoporosis complicated by recurrent bone fractures has been noted as well as conjunctivitis complicated by corneal ulcerations.
All patients have serum IgE levels exceeding 2500 IU/ml. Unlike atopic patients who may also have similarly elevated IgE levels, patients with hyperimmunoglobulin E syndrome have their serum IgE antibody directed to Staph. aureus.112 Usually the patients have normal concentrations of IgG, IgA, and IgM; pronounced blood and sputum eosinophilia; abnormally low anamnestic antibody response; and poor antibody and cell-mediated responses to neoantigens.116 At variable times the neutrophils and monocytes of patients have a profound chemotactic defect. Sera from some, but not all, patients also have been demonstrated to inhibit chemotaxis of normal control neutrophils.
No known therapy is curative, and management decisions are based on the clinical findings. Prophylactic trimethoprim-sulfamethoxazole is effective in reducing infections with Staph. aureus.112 Type and route of antibiotic therapy are dictated by the results of the Gram stain and culture in patients with acute bacterial infections. Incision and drainage are essential for the management of abscesses, including superinfected pneumatoceles. Eczematoid dermatitis can be controlled with topical glucocorticoids to reduce inflammation and antihistamines to control pruritus. Plasmapheresis has been reported to be effective in patients who fail more conservative approaches.
The use of recombinant IFN-a improves the in vitro chemotactic response of neutrophils.117 In five patients treated with recombinant IFNa, their blood mononuclear cells decreased their spontaneous in vitro IgE production with no change in IgG and IgM.118 Clinical trials are now needed to test the efficacy of IFN-g in patients with the syndrome.
Chronic granulomatous disease (CGD) is a genetic disorder affecting 4 to 5 in 1 million humans in which the neutrophils and monocytes ingest but do not kill catalase-positive microorganisms because of an inability to generate antimicrobial oxygen metabolites (see Table 72-1). It is caused by mutations involving one of several genes encoding a component of the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase.119,120
Several laboratory tests are used to classify forms of CGD and aid in understanding its pathogenesis (Table 72-3). The diagnosis is usually made using the nitroblue tetrazolium (NBT) test, in which the yellow water-soluble tetrazolium dye is reduced to a blue insoluble formazan pigment by superoxide anion generated by activated normal phagocytes. Patients with CGD may have a heterogeneous array of symptoms and severity, depending on which subunit is defective and on the nature of the genetic mutation.121


NADPH-Oxidase Function Engulfment of microbes by phagocytic cells is associated with a burst of oxygen consumption that is important for microbicidal killing and digestion. The respiratory burst is accompanied, not by mitochondrial respiration, but by a unique electron transport chain called the NADPH-oxidase (see Chap. 66). Prior to stimulation, the components of the oxidase are physically separated into two major subcellular locations. The membrane-bound portion of the NADPH-oxidase contains a heterodimeric cytochrome b558 composed of a large, heavily glycosylated subunit with a Mr of 91 kD, known as a gp91phox (91-kD glycoprotein of the phagocyte oxidase), and a 22-kD protein known as p22phox.122 The heavy chain of cytochrome b contains sites for heme binding, FAD groups, and NADPH binding.123,124,125 and 126 The three-dimensional structure of cytochrome b558 is not known for certain, but there is a likely cytoplasmic globular domain in the carboxyl terminus half of the peptide that contains consensus sequences for flavin and NADPH binding.127 Current models are also consistent with three transmembrane domains within the amino terminus half of the molecule, which contains the histidines that coordinate heme binding.128 The p22phox also contains a site for heme binding.123 The synthesis of the p22phox peptide is absolutely required for stability of gp91phox and for oxidase activity in the membrane.3 The p22phox contains proline-rich regions that have consensus structure for binding SH3 (SRC homology 3–type domains) found in p47phox.129 Three other proteins vital to the function of this oxidase system have been identified and determined to reside in the cytosol of the resting phagocyte. Upon stimulation, translocation of p47phox takes place. Phosphorylated p47phox together with two other cytoplasmic components of the oxidase, p67phox and a low-molecular-weight guanosine triphosphate rac 2, translocate to the membrane, where they interact with cytoplasmic domains of the transmembrane cytochrome b558 to form the active oxidase.130,131 Both p47phox and p67phox contain two SH3 domains that may participate in intramolecular and intermolecular binding with consensus proline-rich regions in p47phox.132 Phosphorylation, which occurs on serines in the cationic C-terminal region of p47phox, might serve to disrupt this intermolecular interaction, making the SH3 regions available for binding to p22phox. Another cytoplasmic component with homology to p47phox has been identified to be p40phox, which appears to interact with p67phox before and during oxidase assembling.133 An inhibitory role for the p40phox in regulating oxidase activation has been suggested.134
The cell-free system for activating the oxidase has permitted the dissection of the enzyme system into its components and the evaluation of the function of each unit.135,136,137,138,139,140 and 141 Both cytosolic and membrane proteins are required for oxidase activation, and all patients with CGD have defects involving cytochrome b or the cytosolic components p47phox or p67phox.142 The membrane and cytosol interaction for oxidase activation in the cell-free system defines the genetic heterogeneity of CGD.142 Table 72-3 illustrates this point. The neutrophil membrane fractions from patients with XO (X-linked, cytochrome b–negative) and AO (autosomal recessive, cytochrome b–negative) CGD do not support oxidase activation even upon addition of normal cytosol, while the corresponding patient’s cytosols function normally.142 The membrane defect in both these types of CGD is due to the absence of cytochrome b. In the case of A+ CGD (autosomal recessive, cytochrome b–positive), the membrane fraction is normal, whereas the cytosol is severely defective.
Genetic Alterations Affecting Cytochrome b The most frequent form of CGD occurs in two-thirds of patients and is caused by mutations in the gp91phox gene located on chromosome Xp21.1.119,120 These mutations lead to the X-linked form. Large interstitial deletions causing other X-linked disorders, such as retinitis pigmentosa, Duchenne muscular dystrophy, McLeod hemolytic anemia, and ornithine transcarbamylase deficiency, have been reported in a few patients with X-linked CGD.121,143,144 and 145 Mutation analysis of the gene encoding gp91phox and a large group of X-linked CGD kindreds has documented many distinct defects, including point mutations, inversions, deletions, or insertions that disrupt the reading frame and nonsense mutations that create a premature stop codon.119,120,146 Some splice site defects have also been identified. In this situation, short deletions in gp91phox mRNA are caused by point mutations that produce partial or complete exon skipping during mRNA splicing.147 This abnormality is a common cause of X-linked CGD. In the remaining patients, point mutations have been identified that generate either premature stop codons or amino acid substitutions that apparently disrupt protein stability or function and lead to a complete lack of detectable cytochrome b558 protein in phagocytic cells in most patients with X-linked CGD.148 In some situations, low levels of functional cytochrome b are present, whereas in others, normal levels of dysfunctional cytochrome b558 occur.148 In the latter situation there is some clustering of defects in regions of known function, such as the NADPH- or flavin-binding consensus regions.120,146
A similar array of mutations has been identified in the rare CGD patients who have abnormalities in the p22phox gene located on chromosome 16q24.120,149 In this autosomal disorder, mutations in the p22phox gene result in deletions, frameshifts, and/or missense mutations.120 Two patients have been identified as homozygous for missense mutations due to consanguineous heritage. Patients with a defective p22phox gene do not express the other cytochrome unit polypeptide.150 In one patient, a point mutation in p22phox peptide was associated with normal amounts of cytochrome b with normal heme spectrum, but p47phox translocation membrane did not occur and there was no oxidase activation.151 The mutation affected a proline-rich region thought to mediate binding to one of the SH3 domains of p47phox.132 In gp91phox-deficient patients, p22phox mRNA is present, but it is not translated, which is consistent with the notion that either cytochrome subunit polypeptide is dependent upon the stable expression of the other subunit.121
Genetic Alterations Affecting Cytosolic Proteins Two other proteins have been identified as being vital to the function of the NADPH-oxidase system. Their absence results in the syndrome of CGD.152 These proteins have molecular masses of 47 kD and 67 kD, respectively, and are located in the cytosol of resting cells. Defects in the genes for p47phox found on chromosome 7q11 are responsible for one-fourth of all cases of CGD, whereas inherited defects for the gene for neutrophil p67phox account for a small subgroup of autosomal recessive CGD.120 The function of p47phox and p67phox in regulating the respiratory burst oxidase is thought to involve activation of the electron transport function of cytochrome b558. The mutation analysis in patients with p47phox-deficient forms of CGD reveals an unusual pattern, in that more than 90 percent of mutant alleles have guanine-thymine dinucleotide deletion at the start of exon 2, resulting in frameshift and premature stop.120,153 The truncated protein is unstable, in that it cannot be detected immunologically. The majority of patients appear to be homozygous for this mutation without any history of consanguinity.154 The p47phox gene occurs in an area of chromosome 7 that has a high degree of evolutionary duplication in normal individuals because a pseudogene highly homologous to the normal p47phox gene exists in the normal genome in this region of duplication. The pseudogene contains the same GT deletion associated with most cases of p47phox CGD. This implies that recombination of the normal gene and pseudogene with conversion of the normal gene to partial pseudotype sequence in that region may be responsible for the high relative rate of this specific mutation in diverse racial groups.
A second rare form of CGD is caused by mutations in the gene for the p67phox cytosolic component.120 The p67phox gene, which has been mapped to the long arm of chromosome 1, spans 37 kb and contains 16 exons. The mutations identified in p67phox-deficiency CGD have included missense mutations and spliced junction mutations affecting mRNA processing, which led to nondetectable p67phox protein by immunological means.120
The conversion of oxygen to superoxide anion and hydrogen peroxide by the neutrophils is described in Chap. 66. Complex signal transduction pathways serve to link membrane surface receptors with the activation of the respiratory burst oxidase. Cytochrome b is involved in the function of NADPH oxidase.121,141 Cytochrome b has a very low midpoint potential, which makes it thermodynamically feasible for the cytochrome to function as an electron carrier in the oxidase.155 Based on the midpoint potential for cytochrome b, electrons could theoretically be passed from NADPH to a flavin prosthetic group in the oxidase (FAD), then to the heme prosthetic group or cytochrome, and finally to molecular oxygen to form O2–. Indeed, the heavy chain of cytochrome b has been found to bind NADPH and FAD and to contain one of the heme prosthetic groups.127,128 As indicated above, mutations in the gene for cytochrome b558 or the cytosolic factors involved in activating the cytochrome have been associated with the CGD phenotype.
Predisposition to Infection The manner in which the metabolic deficiency of the CGD neutrophil predisposes the host to infection is shown schematically in Fig. 72-3. Normal neutrophils accumulate hydrogen peroxide and other oxygen metabolites in the phagosomes containing ingested microorganisms. Myeloperoxidase is delivered to the phagosome by degranulation, and in this setting hydrogen peroxide acts as a substrate for myeloperoxidase to oxidize halide to hypochlorous acid and chloramines, which kill the microbes. The quantity of hydrogen peroxide produced by the normal neutrophils is sufficient to exceed the capacity of catalase, a hydrogen peroxide-catabolizing enzyme produced by many aerobic microorganisms, including Staph. aureus, most gram-negative enteric bacteria, C. albicans, and Aspergillus spp. In contrast, hydrogen peroxide is not produced by CGD neutrophils, and any generated by the microbes themselves may be destroyed by their own catalase. Thus, catalase-positive microbes can multiply inside CGD neutrophils, where they are protected from most circulating antibiotics, and can be transported to distant sites and released to establish new foci of infection.156 Activation of the oxidase also has a pronounced effect on the pH within the phagocytic vacuole.157 Activation of the respiratory burst is associated with an alkaline phase produced by the pumping of electrons and accompanied by protons along the wall of the phagosome. The alkaline phase is important for the antimicrobial and digestive functions of the neutral hydrolases released from the cytoplasmic granules into the vacuole upon phagocytosis. In CGD, the phagocytic vacuoles remain acidic and the bacteria are not digested properly. In hematoxylin-eosin-stained sections from patients, macrophages may contain a golden pigment, which reflects this abnormal accumulation of ingested material and also contributes to the diffuse granulomata that give CGD its descriptive name.152 On the other hand, when CGD neutrophils ingest pneumococci or streptococci, these organisms generate enough hydrogen peroxide to result in a microbicidal effect.

FIGURE 72-3 The pathogenesis of chronic granulomatous disease. The manner in which the metabolic deficiency of the CGD neutrophil predisposes the host to infection is shown schematically. Normal neutrophils accumulate hydrogen peroxide in the phagosome containing ingested Escherichia coli. Myeloperoxidase is delivered to the phagosome by degranulation, as indicated by the closed circles, and in this setting, hydrogen peroxide acts as a substrate for myeloperoxidase to oxidize halide to hypochlorous acid and chloramines, which kill the microbes. The quantity of hydrogen peroxide produced by the normal neutrophils is sufficient to exceed the capacity of catalase, a hydrogen peroxide–catabolizing enzyme of many aerobic microorganisms, including most gram-negative enteric bacteria, Staph. aureus, C. albicans, and Aspergillus spp. When organisms such as E. coli gain entry into the CGD neutrophils, they are not exposed to hydrogen peroxide because the neutrophils do not produce it, and the hydrogen peroxide generated by microbes themselves is destroyed by their own catalase. When CGD neutrophils ingest streptococci or pneumococci, these organisms generate enough hydrogen peroxide to result in a microbicidal effect. On the other hand, as indicated in the middle figure, catalase-positive microbes, such as E. coli, can survive within the phagosome of the CGD neutrophil.

Although the clinical presentation is variable, several clinical features suggest the diagnosis of CGD.156,158 Any patient with recurrent lymphadenitis should be considered to have CGD. Additionally, patients with bacterial hepatic abscesses, osteomyelitis at multiple sites or in the small bones of the hands and feet, a family history of recurrent infections, or unusual catalase-positive microbial infections all require clinical evaluation for this disorder. The most common clinical disorders that afflict CGD patients are listed in Table 72-4.


The onset of clinical signs and symptoms may occur from early infancy to young adulthood. The attack rate and severity of infections are exceedingly variable. The most common pathogen is Staph. aureus, although any catalase-positive microorganism may be involved. Infection with Serratia marcescens, Pseudomonas cepacia, Aspergillus spp., or C. albicans occurs frequently. Infections are characterized by microabscesses and granuloma formation. The presence of pigmented histiocytes is helpful in establishing the diagnosis. Patients may suffer from the sequelae of chronic infection, including the anemia of chronic disease, lymphadenopathy, hepatosplenomegaly, chronic purulent dermatitis, restrictive lung disease, gingivitis, hydronephrosis, and gastroenteral narrowing. Pneumonias, lymphadenitis, and skin infections are the most common infections encountered. Pneumonias caused by Aspergillus or Nocardia, recurrent lymphadenitis, perirectal abscesses, and recurrent skin infections including folliculitis, cutaneous granulomata, and discoid lupus erythematosus should alert the physician to the possibility of CGD.
Several mothers of patients in whom X-linked inheritance was established had an illness resembling systemic lupus erythematosus.159 Both X-linked and autosomal recessive patients with CGD also have a similar disorder.160,161 It may be that these mothers’ and patients’ cells are unable to clear immune complexes sufficiently, which is a characteristic feature of CGD cells in vitro.162
The defect in the respiratory burst is best determined by measuring superoxide or hydrogen peroxide production in response to both soluble and particulate stimuli. A test that is being employed is the use of flow cytometry using dihydrorhodamine 123 fluorescence.163 Dihydrorhodamine fluorescence detects oxidant production because it increases fluorescence upon oxidation. In most cases there is no detectable superoxide or hydrogen peroxide generation with either type of stimulus. In the variant form of CGD, on the other hand, superoxide may be produced at rates between 0.5 and 10 percent of control.164
An alternative method for measuring respiratory burst activity is the NBT test. This assay is performed by microscopically assessing the ability of individual cells to reduce NBT to purple formazan crystals following stimulation. Commonly there is no NBT reduction with most forms of CGD. In some of the variant forms, however, a high percentage of cells may contain some formazan, a finding indicative of a greatly diminished respiratory burst in most of the neutrophils. This test also permits detection of the carrier state in X-linked CGD when as few as 5 to 10 percent of the cells are NBT-negative.121
More sophisticated procedures can identify the underlying molecular defect. Cytochrome b content can be measured in extracts of detergent-disrupted neutrophils by a spectrophotometric assay.121 Measurement of activity of the patient’s membrane and cytosol in the cell-free oxidase system can be employed along with immunoblotting for a cytochrome b subunit and cytosol oxidase component to characterize X-linked from autosomal recessive forms of CGD. Prenatal diagnosis of CGD is established by performing an NBT test on fetal blood obtained by fetoscopy or by cutaneous umbilical sampling.165 Amniotic fluid cells or chorionic villus biopsy contains fetal DNA that can be employed for earlier prenatal diagnosis of CGD. Restriction fragment length polymorphisms have been successful for diagnosing gp97phox and p67phox deficiency in informative families.166 In other families PCR technology can be employed to analyze fetal DNA if a family’s specific mutation is known.
Leukocytes from patients with CGD have normal glucose-6-phosphate dehydrogenase (G-6-PD) activity. However, a few individuals with apparent CGD have been described that have neutrophils that lack or almost lack in G-6-PD activity.167,168 and 169 The erythrocytes of these patients also lack the enzyme, and the patients have chronic hemolysis. In the cases of severe neutrophil G-6-PD deficiency, an attenuated respiratory burst progressively decreases due to the depletion of intracellular NADPH, the primary substrate for the respiratory burst oxidase. CGD and G-6-PD deficiency can be distinguished from each other by the hemolytic anemia seen in the latter disorder and by the fact that erythrocyte G-6-PD activity is normal in CGD and markedly reduced in G-6-PD deficiency.148
Because marrow transplantation is the only known cure for CGD, vigorous supportive care along with the use of rIFN-g continues to be the foundation of treatment.148,156,170,171 Cultures must be obtained as soon as infection is suspected, as unusual organisms are commonly the source of infection and may grow promptly in vitro. Most abscesses will require surgical drainage for therapeutic and diagnostic purposes, and prolonged use of antibiotics is often required. If fever occurs, it is advisable to obtain certain studies that aid in the management of septic episodes. These include roentgenograms of the chest and skeleton and a CT scan of the liver because of the frequency of pneumonia, osteomyelitis, and liver abscesses.172,173 Arrangements should be made for prompt medical attention at the first signs of infection. With early intervention, many lesions can be managed by conservative medical means. For example, enlarging lymph nodes often regress when treated with local heat and orally administered antistaphylococcal antibiotics. More serious events require hospitalization and a diagnostic and therapeutic approach applicable to any patient with severe infection. In general, antibiotic therapy for the offending organisms is indicated and purulent masses should be drained. The cause of fever and prostration cannot always be established, and empiric treatment with broad-spectrum parenteral antibiotics is required. Often it is necessary to treat with antibiotics for a prolonged time until the initial sedimentation rate approaches normal values.172 Aspergillus spp. infection requires treatment with amphotericin B or, in refractory cases, with granulocyte transfusions.172 Glucocorticoids also may be useful in the treatment of patients with antral and urethral obstruction.174,175 The risk of Aspergillus infection can be reduced by avoiding marijuana smoke and decaying plant material, such as mulch and hay, both of which contain numerous fungal spores.176
Long-term oral prophylaxis with trimethoprim-sulfamethoxazole (5 mg/kg per day of trimethoprim) is an accepted practice in the management of patients with CGD.177 Patients have prolonged infection-free periods, which result from the prevention of infections caused by Staph. aureus, without increasing the incidence of fungal infections. In one series this regimen resulted in a diminished incidence of bacterial infection from 7.1 to 2.4 per one hundred patient-months in patients with autosomal CGD and from 15.8 to 6.9 per hundred patient-months in X-linked patients.177 Use of ketoconazole has not been found to provide any protection against Aspergillus infections, while itraconazole may prove to be efficacious in this regard.178
IFN-g (50 µg/m2, three times per week) can reduce the number of serious infections.179 IFN-g–enhanced neutrophil function in vitro has not been correlated with improvement in the activity of the neutrophil respiratory burst in patients totally lacking the ability to generate superoxide. On the other hand, its use increases the neutrophil expression of the high-affinity Fcg receptor 1 as well as monocyte expression of FcgRI, FcgRII, FcgRIII, CD11/CD18, and HLA-DR.180 The IFN-g protective effect in patients with CGD may involve improved microbial clearance, as suggested by the enhanced phagocytic activity by neutrophils of opsonized Staph. aureus. In rare X-linked CGD patients able to generate some superoxide, IFN-g programs granulocyte cells to increase their expression of cytochrome b, which results in normal superoxide generation.181 With the use of current prophylactic treatments, the mortality in CGD has been reduced to two patient deaths per year per hundred patients followed.3
Mutations in CGD that result in 5 to 10 percent of normal functioning amounts of NADPH have a mild phenotype and better clinical prognosis than that of patients with complete absence of any NADPH oxidase activity.182,183 Similarly, female carriers of X-linked CGD who have only 3 to 5 percent oxidase-normal neutrophils rarely get serious infections suggestive of the CGD clinical phenotype.184 Thus, even low levels or partial correction by gene therapy of CGD are likely to provide clinical benefits. In support of that hypothesis, mouse models of X-linked and p47phox-deficient CGD have been developed by gene targeting.185,186 Studies have shown in the gp91phox- and the p47phox-deficient mouse models of CGD that retrovirus-mediated gene therapy targeting of marrow progenitor cells ex vivo can result in the correction of defects in oxidant production in vivo in peripheral blood neutrophils after radiation conditioning and transplantation of the transduced marrow stem cells.187,188 Protection from infection challenge occurred even when the oxidase-corrected cells comprised less than 10 percent of circulating neutrophils. These promising results have suggested that somatic gene therapy can be employed to correct defective phagocyte oxidase function in selected patients with CGD. In a phase I clinical trial, gene therapy for p47phox-deficiency CGD, five adult patients received intravenous infusions of autologous blood stem cells that were ex vivo transduced using a retrovirus encoding normal p47phox.189 Although conditioning therapy was not given prior to the stem cell infusion, functionally corrected neutrophils were detectable in peripheral blood 3 to 6 weeks after the single infusion and ranged from 0.004 to 0.05 percent of total blood neutrophils. The corrected cells were detectable for as long as 6 months after infusion in some patients. These results indicate a promise for gene therapy in the future to correct phagocytic oxidase function in selected patients with CGD.
The functional and immunochemical absence of the enzyme myeloperoxidase from granules of neutrophils and monocytes, but not eosinophils, is inherited as an autosomal recessive trait, with a prevalence of 1:2000.190 Myeloperoxidase, an enzyme that catalyzes the production of hyperchlorous acid in the phagosome, causes microbicidal deficiency of the neutrophils early after ingestion of microorganisms (see Table 72-1). However, normal microbicidal activity is observed in approximately 1 h after a variety of organisms are ingested.191 Thus, the myeloperoxidase-deficient neutrophil uses a myeloperoxidase-independent system for killing bacteria that is slower than the myeloperoxidase–hydrogen peroxide–halide system but that is eventually effective in eliminating bacteria. Myeloperoxidase-deficient neutrophils accumulate more hydrogen peroxide than do normal neutrophils; the higher peroxide concentration improves the bactericidal activity of the affected neutrophils. In contrast to the retardation of bactericidal activity, candidacidal activity in myeloperoxidase-deficient neutrophils is absent.190,191 The most significant clinical manifestation in a few patients with diabetes mellitus and myeloperoxidase deficiency has been severe infection with C. albicans. Since this is such a common disorder of phagocytes, it is important to note that the vast majority of patients with this genetic disorder have not been unusually susceptible to pyogenic infections and do not require therapy.
The cDNA encoding human myeloperoxidase has been cloned and the gene structure, including promoter and regulatory elements, delineated.3,192,193 The gene consists of 12 exons and 11 introns and is located on the long arm of chromosome 17, and its expression is finely coordinated with expression of genes encoding other lysosomal proteins. Expression of genes for human neutrophil elastase and myeloperoxidase is very similar; it is low in myeloblasts, peaks during the promyelocyte stage, and eventually drops to low levels in myelocytes. Myeloperoxidase is a symmetric molecule composed of four peptides, where each half consists of a heavy- and a light-chain heterodimer.194 Each heavy- and light-chain heterodimer starts as a single peptide that is cleaved during the posttranslational process to yield the heavy and light chains that form half of the mature molecules. The two halves of the molecule are associated by a disulfide linkage between heavy-subunit residues at their residue C319.
The primary translation product of the gene is a single-chain peptide of 80 kD that undergoes cotranslational glycosylation at several asparagine residues, followed by a series of modifications of these oligosaccharides. The apopromyeloperoxidase exists for a prolonged time in the endoplasmic reticulum, where it associates reversibly with several endoplasmic reticulum–resident proteins.195 Subsequent to heme insertion, the enzymatically active promyeloperoxidase undergoes proteolytic cleavage of the pro region. Then, in a prelysosomal compartment, the single peptide is cleaved into the heavy and light subunits, which remain linked. During final sorting within the azurophil lysosome compartment, there is dimerization of half-molecules to form the mature myeloperoxidase.3
Most patients with myeloperoxidase deficiency have a missense mutation in the gene that results in replacement of arginine 569 with tryptophan.196 The mutation results in a precursor that associates with molecular chaperones but does not incorporate heme, resulting in a maturational arrest during processing at the stage of an inactive enzymatic apopromyeloperoxidase. Other patients are compound heterozygotes with one allele bearing the common mutation and the other being normal, resulting in a partial deficiency.3 In one patient, missense mutation resulted in an intact myeloperoxidase molecule that acquired heme but failed to undergo proteolytic processing to a mature molecule.197
There are acquired disorders with associated myeloperoxidase deficiency. Reported states include lead intoxication, ceroid lipofuscinosis, myelodysplastic syndromes, and acute myelogenous leukemia.190 One-half of untreated patients with acute myelogenous leukemia and 20 percent of patients with chronic myelogenous leukemia may have myeloperoxidase deficiency.190,191
Neutrophils contain enzymes capable of inactivating potentially damaging reduced oxygen byproducts. Disposal of superoxide anion is accomplished through superoxide dismutase, a soluble enzyme that converts superoxide to a hydrogen peroxide. Hydrogen peroxide is detoxified by catalase and by the glutathione peroxidase–glutathione reductase system, which converts hydrogen peroxide to water and oxygen.198 In addition to the soluble enzymes, cellular vitamin E serves as an antioxidant to prevent damage to the surface of activated neutrophils when releasing hydrogen peroxide.198 Single cases of profound deficiencies in glutathione reductase199 and glutathione synthetase199 have been associated with impaired neutrophil bactericidal activity (see Table 72-1). Both deficiencies are associated with hemolysis under conditions of oxidative stress (see Chap. 44). Glutathione synthetase deficiency has also been associated with intermittent neutropenia during times of mild infection. Vitamin E has been employed to ameliorate the hemolysis and improve neutrophil function in a patient with glutathione synthetase deficiency.200 Like patients with myeloperoxidase-deficient neutrophils, the patients with glutathione reductase deficiency and glutathione synthetase deficiency are not unusually susceptible to bacterial infections.
An increased susceptibility to pyogenic infections must be viewed in light of a number of factors: (1) adequacy of host defense, (2) the microbes to which the host is exposed, and (3) the conditions of the exposure. It is not always easy to establish a diagnosis of a specific neutrophil dysfunction on clinical grounds alone. Patients with recurrent pyogenic infections often yield no clues as to why they are afflicted, and patients with established deficiency of a defense mechanism may have an unimpressive clinical history. On the other hand, patients may be suspected of having a neutrophil dysfunction if they have a history of frequent bacterial or severe infections. Recurrent pulmonary infections, hepatic abscesses, and perirectal abscesses also should alert the clinician to consider further diagnostic evaluation of neutrophil function. For example, the identification of unusual catalase-positive bacteria and fungi, such as P. cepacia, S. marcescens, Nocardia, and Aspergillus, could be indicative of CGD.
Since many of the tests of neutrophil function are bioassays with great variability, the results of the tests must be interpreted in light of the patient’s clinical condition. For instance, isolated chemotactic defects usually do not explain the propensity for a patient to have recurrent severe infections. Furthermore, variation in bioassays is often intensified by inflammation or infection. An algorithm for evaluation of the patient with recurrent infection is provided in Fig. 72-4.

FIGURE 72-4 Algorithm for the workup patients with recurrent infections. Abbreviations: CBC, complete blood count; G-6-PD, glucose-6-phosphate dehydrogenase; Ig, immunoglobulin; LAD, leukocyte adhesion deficiency. (Modified from Curnutte JT, Boxer LA: Clinically significant phagocytic cell defects, in Current Clinical Topics in Infectious Diseases, 6th ed, edited by JS Remington, MN Swartz, p 144. McGraw-Hill, New York, 1985.)


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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology




  2. *There are certainly a lot of details like that to take into consideration. That is a great point to bring up. I offer the thoughts above as general inspiration but clearly there are questions like the one you bring up where the most important thing will be working in honest good faith. I don?t know if best practices have emerged around things like that, but I am sure that your job is clearly identified as a fair game. Both boys and girls feel the impact of just a moment’s pleasure, for the rest of their lives.

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